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Introduction


RNase A is an endonuclease that cleaves and breaks down RNA using acid base catalysis. RNase A has been a model protein for studies on the stability, folding and chemistry of proteins. ‘ ’ It is also essential in protein regulation within the body due to its function of RNA degradation.

Substrate Binding


To determine the structural characteristics of RNA substrate binding to RNase A, X-ray crystallography was used to image inhibitory DNA tetramers bound to RNase A. DNA lacks the 2’OH essential to RNA cleavage, making the complex more conducive to crystallography. The complex between RNase A and thymidylic acid tetramer (d(pT)4) (1rta) provides information about specificity of the binding pocket subunits, B0, B1, B2 and B3. Many interactions observed in this complex occur between amino acid residues and the nucleic acid backbone. Examples of these interactions include hydrogen bonding between  phosphate of T1 and Arg39 as well as hydrogen bonding between the O5’ oxygen of the ribose of T3 and Lys41. ‘ ’



Further binding pocket characterization was performed using RNase A complexed with the oligonucleotide d(ApTpApApG) (1rcn). This tetramer was important in determining the specificity of the binding sites of RNase A. In this complex, the B1 site is thought to exclusively bind to pyrimidine bases due to steric interactions and hydrogen bonding to Thr45d. When Thr45 was mutated to glycine, purines readily bound to the B1 site. ‘ ’ This interaction appears to be the driving force behind its inability to bind purines. While binding of other nucleobases to the B2 and B3 sites is possible, the imaging of this complex elucidated the preferences for adenosine bases at these two positions. In addition to its catalytic activity His119 has also been shown to be important in substrate specificity. This is due to the pi stacking between His119 and A3. When this site was mutated, the affinity for a poly(A) substrate was decreased by 104-fold. ‘ It also establishes hydrogen bonding between Asn71-A3, Gln69-A3 and Gln69-A4. ‘ ’

Conclusion
From the studies on RNase A complexes with deoxy nucleic acid tetramers, it has been established that this enzyme recognizes the substrate on both its phosphate backbone and on individual nucleobases. RNase A has a nonspecific B0 site, a B1 site specific to pyrimidines and a B3 and B4 site with a preference for adenosine bases. Similar to other enzymes, RNase A uses the hydrogen bonding distance between amino acids and the substrate to bind specifically to certain nucleobases. Studying the substrate recognition and specificity of enzymes such as RNase A is an important step in understanding the regulation of RNA within biological systems.

External Resources

 * Full crystal structure of 1RTA
 * Full crystal structure of 1RCN
 * Further information on 1RTA
 * RNase A- Molecule of the Month